Towards Sustainable Medicinal Resources through Marine Soft Coral Aquaculture: Insights into the Chemical Diversity and the Biological Potential

In recent decades, aquaculture techniques for soft corals have made remarkable progress in terms of conditions and productivity. Researchers have been able to obtain larger quantities of soft corals, thus larger quantities of biologically active metabolites, allowing them to study their biological activity in many pharmacological assays and even produce sufficient quantities for clinical trials. In this review, we summarize 201 secondary metabolites that have been identified from cultured soft corals in the era from 2002 to September 2022. Various types of diterpenes (eunicellins, cembranes, spatanes, norcembranes, briaranes, and aquarianes), as well as biscembranes, sterols, and quinones were discovered and subjected to bioactivity investigations in 53 different studies. We also introduce a more in-depth discussion of the potential biological effects (anti-cancer, anti-inflammatory, and anti-microbial) and the mechanisms of action of the identified secondary metabolites. We hope this review will shed light on the untapped potential applications of aquaculture to produce valuable secondary metabolites to tackle current and emerging health conditions.


Aquaculture Background
Nature has contributed to all aspects of drug discovery and development through the production of secondary metabolites with diverse chemical structures and biological activities. In addition to terrestrial plants and animals, marine organisms are considered a prolific resource of highly potent compounds. Although occupying 70% of the earth surface, only less than 5% of deep-sea organisms have been studied [1], which opens an enormous opportunity for marine drug discovery, especially with the assistance of newly developed specialized techniques and equipment. Among marine organisms, soft corals are marine animals that belong to the order Alcyonacea. Thousands of secondary metabolites have been isolated from soft corals with a wide range of biological activities, such as antibacterial, antitumor, anti-inflammatory, antifungal, antiviral, antioxidant, antiallergic, and antimalarial activities [2]. However, the interesting compounds are usually extracted in very low amounts. Once a compound is isolated and identified as a potent therapeutic agent by in vitro or in vivo assays, a kilogram-scale amount could be required for further gest difference between our current work and the previous reviews.
In this review, we aim to provide a general summary of the chemical diversity of natural products derived from cultured soft corals and their potential bioactivities ( Figure  1). Different keywords, such as "cultured", "soft corals", and "secondary metabolites", were searched in various platforms, including Pubmed, Google Scholar, ScienceDirect, ResearchGate, and Reaxys. A total of 86 references with 201 derivatives were found to be related to the target topic and discussed throughout the current review. We try in this work to highlight that the aquaculture of these marine organisms can provide a consistent, reproducible, and sustainable source of potent marine compounds, aiding in the discovery and development of new drugs from both known and novel bioactive secondary metabolites.

Eunicellin-Based Diterpene
Eunicellin-based diterpenes (Figures 2 and 3) are marine natural products that consist of a six-membered ring moiety fused to a ten-membered ring moiety. In many compounds, an additional ether bridge is formed between C-2 and C-9 or C-4 and C-7 [23].
The investigation of new compounds from the cultured soft coral Klyxum simplex led to the isolation and identification of 27 novel secondary metabolites, including klysimplexins A-X (compounds 1-24) [24][25][26] and klysimplexin sulfoxides A-C (compounds 25-
Dihydrosinularin (46), a cembranoid derivative, was also obtained from the cultured Sinularia flexibilis. The compound was further tested for its anti-inflammatory activity and showed no inhibitory effects on the NO expression due to a slight difference with active derivative. Hydrogenation at C-17 formed a single bond in 46 instead of a double bond as in sinularin [33].
The investigation of new compounds from the cultured soft coral Klyxum simplex led to the isolation and identification of 27 novel secondary metabolites, including klysimplexins A-X (compounds 1-24) [24][25][26] and klysimplexin sulfoxides A-C (compounds 25-27) [27]. Most of the compounds were obtained as a colorless oil, except for klysimplexins A, which consisted of colorless crystals following recrystallization from acetone. Their molecular formulas were established based on their HRESIMS spectra and 13 C NMR data. The IR spectra of klysimplexin sulfoxides showed that they not only possess hydroxy, carbonyl, and ester functionalities as revealed in the spectra of klysimplexins but also contain sulfoxide groups in their structure. In general, these compounds share a common eunicellin-based skeleton which was partially elucidated with the assistance of IR, 1D, and 2D NMR spectroscopic data. Single-crystal X-ray diffraction analysis was exploited to establish the detailed structure and relative configuration of klysimplexin A (1). The absolute configurations of klysimplexins A (1), C (3), L (12), and V (22) were further determined by the modified Mosher's method [24][25][26]. The NMR spectroscopic data confirmed that the eunicellinebased structure of klysimplexin I (9) was similar to a known compound, simplexin B, which was isolated from the same species. The replacement of a hydroxy group at C-6 in simplexin B by a myristate moiety at C-6 in 9 was confirmed by a base-catalyzed hydrolysis of 9, which afforded simplexin B after the reaction [25]. The authors indicated that klysimplexin P (15) is a 6,7-secoeunicellin while klysimplexin T (20) possesses a tricarbocyclic skeleton, which might be derived from the carbon-carbon bond formation between C-2 and C-7 of the corresponding 2,9-deoxygenated eunicellin. Among these novel metabolites, klysimplexins U (21) and V (22) were reported as the first 4-oxygenated eunicellin-based diterpenes that were isolated from the genera Klyxum [26], and klysimplexin sulfoxides A-C (compounds 25-27) were the first discovered sulfoxide-containing eunicellin-type derivatives [27] (Figure 3).
A new cembranoid, trocheliophorol A (92), was isolated from the cultured soft coral Sarcophyton trocheliophorum. Based on the findings from an extensive analysis of the spectral data of 92, it was demonstrated that the novel compound was obtained as the first example of an α,β-unsaturated-γ-lactone cembrane possessing a tetrahydrofuran moiety with a rare 8,11-ether linkage [39].
Three new furanocembranoids-briaviodiol F (93) and briaviotriols A (94) and B (95), along with a known analogue, briaviodiol A (96), were discovered from the cultured-type Briareum violaceum. The structures of the novel compounds were determined as tricyclic cembrane diterpenes possessing tetrahydrofuran rings by the interpretation of their An investigation of the chemical constituents of the cultured soft coral Sinularia flexibilis led to the purification of three cembranoids, including flexibilisolide A (31), flexibilisin A (32), and 11-epi-sinulariolide acetate (33) [29]. 11-epi-Sinulariolide acetate was a known compound that was originally isolated and identified from the wild soft corals Sinularia notanda and Sinularia querciformis collected from the Gulf of Elat [30]. The comparison of NMR data of 32 with those of 33 showed a similarity in the structure, except for an additional methoxy group that was observed in 32. This was confirmed by a base-catalyzed hydrolysis of 33 that afforded 32 as a product of the reaction [29].
Flexibilide (34), also known as sinularin, was first isolated from the soft coral Sinularia flexibilis collected from the Hayman Island of Australia. Its structure was established by NMR and X-ray crystallography. The anti-inflammatory and analgesic effects of flexibilide were performed using a material isolated from the same species that was cultured in a cultivation tank of the National Museum of Marine Biology and Aquarium in Taiwan. The purity of flexibilide was identified and verified by 1 H and 13 C NMR spectra before the in vivo study [31].
Mar. Drugs 2022, 20, x 9 of 31 spectroscopic data. The known metabolite 96 was identified as briaviodiol A through the comparison of its 1 H and 13 C NMR spectra with the published data [40]. A NMR-directed investigation of chemical constituents from the cultured octocoral Briareum violaceum led to the discovery of four new hydroperoxyfurancembranoids, briaviodiols B-E (97-100) [41].  Dihydrosinularin (46), a cembranoid derivative, was also obtained from the cultured Sinularia flexibilis. The compound was further tested for its anti-inflammatory activity and showed no inhibitory effects on the NO expression due to a slight difference with active derivative. Hydrogenation at C-17 formed a single bond in 46 instead of a double bond as in sinularin [33].
A new cembranoid, trocheliophorol A (92), was isolated from the cultured soft coral Sarcophyton trocheliophorum. Based on the findings from an extensive analysis of the spectral data of 92, it was demonstrated that the novel compound was obtained as the first example of an α,β-unsaturated-γ-lactone cembrane possessing a tetrahydrofuran moiety with a rare 8,11-ether linkage [39].
Three new furanocembranoids-briaviodiol F (93) and briaviotriols A (94) and B (95), along with a known analogue, briaviodiol A (96), were discovered from the culturedtype Briareum violaceum. The structures of the novel compounds were determined as tricyclic cembrane diterpenes possessing tetrahydrofuran rings by the interpretation of their spectroscopic data. The known metabolite 96 was identified as briaviodiol A through the comparison of its 1 H and 13 C NMR spectra with the published data [40].
A NMR-directed investigation of chemical constituents from the cultured octocoral Briareum violaceum led to the discovery of four new hydroperoxyfurancembranoids, briaviodiols B-E (97-100) [41].
Leptoclalin A (101) (Figure 7) was isolated as a colorless oil from cultured soft coral Sinularia leptoclados. Its molecular formula was determined as C 20 H 32 O based on its HRESIMS spectrum. The detailed analysis of its spectroscopic data, in particular 2D NMR, resulted in the establishment of a new rare spatane diterpenoid [43]. Spatane-type diterpenes are tricyclic terpenoids derived from a prenylgermacrane by 1,5-and 6,10-cyclisation [42].
Leptoclalin A (101) (Figure 7) was isolated as a colorless oil from cultured soft coral Sinularia leptoclados. Its molecular formula was determined as C20H32O based on its HRESIMS spectrum. The detailed analysis of its spectroscopic data, in particular 2D NMR, resulted in the establishment of a new rare spatane diterpenoid [43].

Briarane-Type Diterpene
Briarane-type diterpenes (Figures 9-11) are derived from the 3,8-cyclization of cembranoid [46]. This type of diterpenoid skeleton is only found in marine organisms, particularly in octocorals [47]. The chemical investigation of the cultured scleraxonia Briareum stechei led to the discovery of four new diterpenoids-briaexcavatins I-L (105-108). Other two known compounds, excavatolides C (109) and E (110), were first found in the wildtype Briareum stechei discovered in Taiwan and were also isolated from the culture-type coral. The absolute configurations of 109 and 110 were further confirmed by X-ray data analysis for the first time [48].
Guided by the proton NMR signals for interesting structures, the ethyl acetate layer that was obtained by partitioning the organic extract of the cultured Briareum stechei afforded four new briarane-related diterpenes. They were assigned as briaexcavatins M-P (111-114) based on the analysis of extensive spectroscopic data [49]. Subsequently, briaexcavatins Q-T (115-118) were isolated from the same cultured species [50].
A new chlorinated briarane, briaexcavatin U (119), was also isolated from a cultured octocoral Briareum stechei. After isolation as a white powder, the compound was
Leptoclalin A (101) (Figure 7) was isolated as a colorless oil from cultured soft coral Sinularia leptoclados. Its molecular formula was determined as C20H32O based on its HRESIMS spectrum. The detailed analysis of its spectroscopic data, in particular 2D NMR, resulted in the establishment of a new rare spatane diterpenoid [43].

Briarane-Type Diterpene
Briarane-type diterpenes (Figures 9-11) are derived from the 3,8-cyclization of cembranoid [46]. This type of diterpenoid skeleton is only found in marine organisms, particularly in octocorals [47]. The chemical investigation of the cultured scleraxonia Briareum stechei led to the discovery of four new diterpenoids-briaexcavatins I-L (105-108). Other two known compounds, excavatolides C (109) and E (110), were first found in the wildtype Briareum stechei discovered in Taiwan and were also isolated from the culture-type coral. The absolute configurations of 109 and 110 were further confirmed by X-ray data analysis for the first time [48].
Guided by the proton NMR signals for interesting structures, the ethyl acetate layer that was obtained by partitioning the organic extract of the cultured Briareum stechei afforded four new briarane-related diterpenes. They were assigned as briaexcavatins M-P (111-114) based on the analysis of extensive spectroscopic data [49]. Subsequently, briaexcavatins Q-T (115-118) were isolated from the same cultured species [50].
A new chlorinated briarane, briaexcavatin U (119), was also isolated from a cultured octocoral Briareum stechei. After isolation as a white powder, the compound was

Briarane-Type Diterpene
Briarane-type diterpenes (Figures 9-11) are derived from the 3,8-cyclization of cembranoid [46]. This type of diterpenoid skeleton is only found in marine organisms, particularly in octocorals [47]. The chemical investigation of the cultured scleraxonia Briareum stechei led to the discovery of four new diterpenoids-briaexcavatins I-L (105-108). Other two known compounds, excavatolides C (109) and E (110), were first found in the wild-type Briareum stechei discovered in Taiwan and were also isolated from the culture-type coral. The absolute configurations of 109 and 110 were further confirmed by X-ray data analysis for the first time [48].
Guided by the proton NMR signals for interesting structures, the ethyl acetate layer that was obtained by partitioning the organic extract of the cultured Briareum stechei afforded four new briarane-related diterpenes. They were assigned as briaexcavatins M-P (111-114) based on the analysis of extensive spectroscopic data [49]. Subsequently, briaexcavatins Q-T (115-118) were isolated from the same cultured species [50].
A new chlorinated briarane, briaexcavatin U (119), was also isolated from a cultured octocoral Briareum stechei. After isolation as a white powder, the compound was recrystallized as colorless prisms for further X-ray analysis to establish its absolute stereochemistry [51].    In a recent study on the chemical constituents of the cultured soft coral Briareum violaceum, three new polyacetoxybriarane diterpenoids, briavioids A-C (166-168), as well as two known analogues, briaexcavatin M (111) and excavatolide F (169), were isolated. The structure of briavioid A (166) was fully confirmed by single-crystal X-ray diffraction analysis [65] (Figure 11).

Aquariane-Type Diterpene
Aquariane-type diterpenes possess a tricyclic system that includes a nine-membered ring fused to two five-membered rings [66]. The aquariane scaffold is assumed to be a biosynthetic product of a precursor possessing briarane skeleton by the sequent di-π-methane and vinyl-cyclopropane rearrangements. Aquariolide A (170), a new compound isolated from the cultured Erythropodium caribaeorum, was an example of an aquariane skeleton [28] (Figure 12).

Biscembranoid
Biscembranoids are a group of tetraterpenoids that possess a 14-6-14-membered tricyclic system [14]. Commonly, they are biosynthesized by the Diels-Alder reaction of two monocembranoidal units. This type of marine-derived metabolite has been mainly found in the soft corals belonging to the genera Sarcophyton, Lobophytum, and Sinularia. A total of eight biscembranes were isolated from the cultured soft corals of the genera Sarcophyton [8,38] (Figure 13).
Two structurally novel biscembranoids, glaucumolides A (171) and B (172), along with a known biscembrane, ximaolide A (173), were isolated from the cultured soft coral Sarcophyton glaucum. The structures and absolute configurations of the new metabolites were established using extensive spectroscopic analyses, including a comparison of their circular dichroism spectroscopic data with those of the related compounds. All evidence from the spectroscopic data demonstrated that the two new biscembranes possess an unprecedented molecular scaffold that is biosynthesized by the Diels-Alder reaction between isosarcophytonolide D (74) and a novel diene monomer precursor, ε-lactonecembrane [38]. Figure 11. Newly isolated briaranes from the cultured soft coral Briareum violaceum (166-169).
Five new briarane derivatives, briaexcavatins V-Z (120-124), were isolated from the cultured octocoral Briareum stechei. The absolute stereochemistry of briaexcavatin X (121) was confirmed on the basis of single-crystal X-ray diffraction data. In addition to the structure elucidation, the relationships between 13 C NMR chemical shifts and the conformations of briaranes possessing an 11,12-epoxy group were also discussed. Among the five novel compounds, briaexcavatin Y (123) was the first example of a briarane possessing a C-8/9 epoxy group [52].
The long-term study of Briareum stechei chemical constituents led to the isolation of two novel briarane derivatives, excavatoids A (125) and B (126), and a known metabolite, briaexcavatin I (105). Compounds 125 and 105 were subjected to a single-crystal X-ray diffraction analysis to establish their absolute configurations. Among these isolates, excavatoid A (125) was reported as the first briarane, possessing six hydroxy groups and a 17-methoxy group [53].
Two new metabolites that possess a briarane skeleton were obtained by the analysis of chemical constituents of the cultured octocoral Briareum stechei. The identified compounds were designated as briarenols O (150) and P (151), in which briarenol O (150) was believed to be the second analogue that possesses a rare 2-ketobriarane skeleton. At the same time, a known analogue, excavatolide C (109), was also isolated along with the novel derivatives [46].
Four new compounds, briarenols Q-T (152-155), were obtained from the organic extract of the cultured octocoral Briareum stechei using high performance liquid chromatographic approaches [63].
Eight chlorinated briarane diterpenoids were purified from the organic extract of the cultured-type Briareum stechei. The structure of four previously unreported metabolites, briarenols W-Z (156-159), were established using spectroscopic analyses, while the known ones were identified as solenolide A (160), briarenolide M (161), briaexcavatolide F (162), and brianolide (163) based on the comparison of their spectroscopic data with literature values. The absolute configuration of 163 was also determined using single-crystal X-ray diffraction analysis [64]. The methanol extract of the cultured Erythropodium caribaeorum afforded two briaranes, erythrolides A (164) and B (165), which were also found in the wild type of the same species [28].
In a recent study on the chemical constituents of the cultured soft coral Briareum violaceum, three new polyacetoxybriarane diterpenoids, briavioids A-C (166-168), as well as two known analogues, briaexcavatin M (111) and excavatolide F (169), were isolated. The structure of briavioid A (166) was fully confirmed by single-crystal X-ray diffraction analysis [65] (Figure 11).

Aquariane-Type Diterpene
Aquariane-type diterpenes possess a tricyclic system that includes a nine-membered ring fused to two five-membered rings [66]. The aquariane scaffold is assumed to be a biosynthetic product of a precursor possessing briarane skeleton by the sequent di-πmethane and vinyl-cyclopropane rearrangements. Aquariolide A (170), a new compound isolated from the cultured Erythropodium caribaeorum, was an example of an aquariane skeleton [28] (Figure 12).
In a recent study on the chemical constituents of the cultured soft coral Briareum violaceum, three new polyacetoxybriarane diterpenoids, briavioids A-C (166-168), as well as two known analogues, briaexcavatin M (111) and excavatolide F (169), were isolated. The structure of briavioid A (166) was fully confirmed by single-crystal X-ray diffraction analysis [65] (Figure 11).

Aquariane-Type Diterpene
Aquariane-type diterpenes possess a tricyclic system that includes a nine-membered ring fused to two five-membered rings [66]. The aquariane scaffold is assumed to be a biosynthetic product of a precursor possessing briarane skeleton by the sequent di-π-methane and vinyl-cyclopropane rearrangements. Aquariolide A (170), a new compound isolated from the cultured Erythropodium caribaeorum, was an example of an aquariane skeleton [28] (Figure 12).

Biscembranoid
Biscembranoids are a group of tetraterpenoids that possess a 14-6-14-membered tricyclic system [14]. Commonly, they are biosynthesized by the Diels-Alder reaction of two monocembranoidal units. This type of marine-derived metabolite has been mainly found in the soft corals belonging to the genera Sarcophyton, Lobophytum, and Sinularia. A total of eight biscembranes were isolated from the cultured soft corals of the genera Sarcophyton [8,38] (Figure 13).
Two structurally novel biscembranoids, glaucumolides A (171) and B (172), along with a known biscembrane, ximaolide A (173), were isolated from the cultured soft coral Sarcophyton glaucum. The structures and absolute configurations of the new metabolites were established using extensive spectroscopic analyses, including a comparison of their circular dichroism spectroscopic data with those of the related compounds. All evidence from the spectroscopic data demonstrated that the two new biscembranes possess an unprecedented molecular scaffold that is biosynthesized by the Diels-Alder reaction between isosarcophytonolide D (74) and a novel diene monomer precursor, ε-lactonecembrane [38].

Biscembranoid
Biscembranoids are a group of tetraterpenoids that possess a 14-6-14-membered tricyclic system [14]. Commonly, they are biosynthesized by the Diels-Alder reaction of two monocembranoidal units. This type of marine-derived metabolite has been mainly found in the soft corals belonging to the genera Sarcophyton, Lobophytum, and Sinularia. A total of eight biscembranes were isolated from the cultured soft corals of the genera Sarcophyton [8,38] (Figure 13).
Two structurally novel biscembranoids, glaucumolides A (171) and B (172), along with a known biscembrane, ximaolide A (173), were isolated from the cultured soft coral Sarcophyton glaucum. The structures and absolute configurations of the new metabolites were established using extensive spectroscopic analyses, including a comparison of their circular dichroism spectroscopic data with those of the related compounds. All evidence from the spectroscopic data demonstrated that the two new biscembranes possess an unprecedented molecular scaffold that is biosynthesized by the Diels-Alder reaction between isosarcophytonolide D (74) and a novel diene monomer precursor, ε-lactonecembrane [38].
Four new biscembranes, sardigitolides A-D (174-177), were isolated from the culturedtype Sarcophyton digitatum. At the same time, three previously known biscembranoidal derivatives were also purified from the cultivated soft coral and were identified as sarcophytolide L (178) and glaucumolides A (171) and B (172) [8]. Four new biscembranes, sardigitolides A-D (174-177), were isolated from the cultured-type Sarcophyton digitatum. At the same time, three previously known biscembranoidal derivatives were also purified from the cultivated soft coral and were identified as sarcophytolide L (178) and glaucumolides A (171) and B (172) [8].

Steroid
Different types of steroids were obtained from Litophyton columnaris and the genera Sinularia, including four sterols and sixteen withanolidal and non-withanolidal steroids ( Figure 14).
A chemical investigation demonstrated that the cultured-type soft coral Sinularia brassica is an abundant supply of bioactive steroids as compared with the wild type of the same species. A series of sixteen new withanolidal and non-withanolidal steroids, including sinubrasolides A-G (183-189) [68], sinubrasolides H-L (190-194) [

Steroid
Different types of steroids were obtained from Litophyton columnaris and the genera Sinularia, including four sterols and sixteen withanolidal and non-withanolidal steroids ( Figure 14).
A chemical investigation demonstrated that the cultured-type soft coral Sinularia brassica is an abundant supply of bioactive steroids as compared with the wild type of the same species. A series of sixteen new withanolidal and non-withanolidal steroids, including sinubrasolides A-G (183-189) [68], sinubrasolides H-L (190-194) [69], and sinubrasones A-D (195)(196)(197)(198) [70], were purified and identified from the soft coral Sinularia brassica cultivated in a tank for five years. The authors also mentioned that sinubrasolides H, I, and K (190, 191, and 193) shared a common 16,23-oxa-bridged tetrahydropyran framework, which was considered as a novel structure of withanolidal steroid [69].  (195)(196)(197)(198) [70], were purified and identified from the soft coral Sinularia brassica cultivated in a tank for five years. The authors also mentioned that sinubrasolides H, I, and K (190, 191, and 193) shared a common 16,23-oxa-bridged tetrahydropyran framework, which was considered as a novel structure of withanolidal steroid [69].

Miscellaneous
In addition to the aforementioned groups, several compounds that contributed to a minor portion of nearly 200 marine metabolites were purified from different species of cultured soft corals, such as a quinone, a hydroquinone, and an α-tocopherol derivative.

Miscellaneous
In addition to the aforementioned groups, several compounds that contributed to minor portion of nearly 200 marine metabolites were purified from different species cultured soft corals, such as a quinone, a hydroquinone, and an α-tocopherol derivative A new quinone derivative ( Figure 14) was isolated from the cultured soft coral Sin laria flexibilis and was designated as flexibilisquinone (199) [71]. The organic extract of th cultivated Sarcophyton tenuispiculatum yielded a new 1,4-dihydrobenzoquinone, name sarcotenuhydroquinone (200) [9]. The ethyl acetate extract of the cultured Lobophytum cra sum afforded a new α-tocopherol derivative, crassumtocopherol C (201) (Figure 15) [7].

Anti-Cancer Activity
The cytotoxicity of a series of new eunicelline-based diterpenes isolated from the cu tured soft coral Klyxum simplex was evaluated against six human tumor cell lines, includ ing human liver carcinoma (Hep G2 and Hep G3B), human breast carcinoma (MDA-MB 231 and MCF-7), human lung carcinoma (A-549), and human oral cancer cells (Ca9- 22 The results showed that only klysimplexins B (2) and H (8) exhibited a significant cyt toxic effect against a limited panel of cancer cell lines, in which klysimplexin B (2) wa more potent against Hep G2, Hep 3B, MDA-MB-231, MCF-7, A549, and Ca9-22 cell line It was believed that the potent cytotoxic effect of klysimplexin B (2) against different tu mor cell lines might be caused by the distinguished α,β-unsaturated ketone in its structu when compared with that of the other analogues [24][25][26].
Cembranes are the most popular secondary metabolites that have been found in th chemical investigation of cultured soft corals. Among those, half the isolates displaye anti-cancer effects on various cell lines, contributing to nearly 50% anti-cancer com pounds. 11-epi-Sinulariolide acetate (33) and 11-dehydrosinulariolide (41) are the two typ ical cembranoids that have been subjected to significant anti-cancer research. 11-epi-Sinu lariolide acetate (33), a cembrane isolated from the cultured soft coral Sinularia flexibili exhibited weak cytotoxicity against the proliferation of MCF-7 cells with ED50 of 11 μg/mL [29]. However, the compound showed a significant anti-proliferative effect, alon with an inhibitory activity on cell migration and invasion, against hepatocellular carc noma cells (HA22T) in a concentration-dependent manner. The molecular mechanism anti-metastasis effects of 33 was further clarified by Western blot analysis, which sug gested that the cytotoxic effect of this compound was mediated through ERK1/ p38MAPK, and FAK/PI3K/AKT/mTOR signaling pathways [72]. Another study showe that the anti-proliferative effect of 33 on HA22T cells was apoptosis mediated throug mitochondrial dysfunction and endoplasmic reticulum stress-induced pathways [73

Anti-Cancer Activity
The cytotoxicity of a series of new eunicelline-based diterpenes isolated from the cultured soft coral Klyxum simplex was evaluated against six human tumor cell lines, including human liver carcinoma (Hep G2 and Hep G3B), human breast carcinoma (MDA-MB-231 and MCF-7), human lung carcinoma (A-549), and human oral cancer cells (Ca9-22). The results showed that only klysimplexins B (2) and H (8) exhibited a significant cytotoxic effect against a limited panel of cancer cell lines, in which klysimplexin B (2) was more potent against Hep G2, Hep 3B, MDA-MB-231, MCF-7, A549, and Ca9-22 cell lines. It was believed that the potent cytotoxic effect of klysimplexin B (2) against different tumor cell lines might be caused by the distinguished α,β-unsaturated ketone in its structure when compared with that of the other analogues [24][25][26].
Cembranes are the most popular secondary metabolites that have been found in the chemical investigation of cultured soft corals. Among those, half the isolates displayed anti-cancer effects on various cell lines, contributing to nearly 50% anti-cancer compounds. 11-epi-Sinulariolide acetate (33) and 11-dehydrosinulariolide (41) are the two typical cembranoids that have been subjected to significant anti-cancer research. 11-epi-Sinulariolide acetate (33), a cembrane isolated from the cultured soft coral Sinularia flexibilis, exhibited weak cytotoxicity against the proliferation of MCF-7 cells with ED 50 of 11.5 µg/mL [29]. However, the compound showed a significant anti-proliferative effect, along with an inhibitory activity on cell migration and invasion, against hepatocellular carcinoma cells (HA22T) in a concentration-dependent manner. The molecular mechanism of anti-metastasis effects of 33 was further clarified by Western blot analysis, which suggested that the cytotoxic effect of this compound was mediated through ERK1/2, p38MAPK, and FAK/PI3K/AKT/mTOR signaling pathways [72]. Another study showed that the anti-proliferative effect of 33 on HA22T cells was apoptosis mediated through mitochondrial dysfunction and endoplasmic reticulum stress-induced pathways [73]. Based on the results, the authors suggested that 33 could be a promising candidate to inhibit metastasis, prevent invasion, and treat hepatocellular carcinoma.
The anti-tumor effect of a cembranoid derivative, 11-dehydrosinulariolide (41), on oral cancer cells (CAL-27 and Ca9-22) was thoroughly evaluated by MTT assay, flow cytometry, and wound-healing assay. A comparative proteomic analysis was also performed to compare the expression of overall protein expression in cancer cells treated with 11-dehydrosinulariolide (41) and in the control cells. The results showed that 11dehydrosinulariolide (41) reduced the cell viability to 70% and significantly induced both the early and late apoptosis of CAL-27 cells at a concentration of 1.5 µg/mL. Similarly, at a dose of 3.0 µg/mL, the cell viability of Ca9-22 was also reduced to 60%, and the apoptosis of Ca9-22 cells was significantly induced by 11-dehydrosinulariolide (41). Additionally, the cell migration of the two cell lines was inhibited in a concentration-dependent manner. The levels of differential proteins related to the apoptosis or inhibition of cancer cell growth were also regulated by 11-dehydrosinulariolide (41) in both cell lines [74,75]. In another study, 11-dehydrosinulariolide (41) exhibited dose-dependent cytotoxicity against A2058 melanoma cells with the IC 50 of 5.8 µg/mL. The anti-migratory activity of the compound was investigated with doses ranging from 2 to 6 µg/mL, demonstrating suppression rates of 32%, 51%, and 73% for 2, 4, and 6 µg/mL of 11-dehydrosinulariolide, respectively [76]. The anti-tumor effects of 11-dehydrosinulariolide against human small cell lung cancer cells were also demonstrated in vitro and in vivo experiments [77]. The revealed evidence proved that 11-dehydrosinulariolide is a promising therapeutic agent to be developed as an anti-cancer drug.
Four novel non-withanolidal steroids, sinubrasones A-D (195)(196)(197)(198), were evaluated for their cytotoxicity against a limited panel of cancer cell lines. The authors showed that sinubrasones B and C (196 and 197) were more cytotoxic than the other two compounds, and the potent was activity attributed to the presence of a methyl ester at C-25 [70].
The effect of flexibilide (34) on the expression of pro-inflammatory proteins, iNOS and COX-2, and anti-inflammatory transforming growth factor-β (TGF-β) in RAW 264.7 cells induced by lipopolysaccharide was evaluated using Western blot analysis. The results revealed its anti-inflammatory activity through the significant changes in protein levels at 10 µM and 20 µM resulting in the inhibition of iNOS and COX-2, along with the upregulation of TGF-β [80].
Briaranes are the diterpene group that possesses the highest number of anti-inflammatory metabolites among the isolates of cultured soft corals. Out of 30 compounds, 21 displayed significantly inhibitory effects on superoxide anion generation and elastase release by human neutrophils, as well as the levels of iNOS and COX-2. The most remarkable bioactive compounds are four new briaranes, including excavatoids I (131) and L (134), and briaviolides L (142) and O (145), exhibiting 38.3%, 42.44%, 38.19%, and 89.47% inhibitory effects on elastase release, superoxide anion generation, and the levels of COX-2 and iNOS, respectively [55,56,58,59].
The three novel biscembranoids isolated from Sarcophyton spp., glaucumolides A (171), B (172), and ximaolide A (173), exhibited their great inhibitory effect levels of superoxide anion generation and elastase release, as well as the levels of proinflammatory iNOS and COX-2 proteins. The results showed that glaucumolide A (171) inhibited superoxide anion generation and elastase release in human neutrophils with the IC 50 values of 2.79 ± 0.66 and 3.97 ± 0.10 µM, respectively. It also significantly reduced the levels of iNOS and COX-2 to −2.6 ± 2.7 and −0.5 ± 3.2% at 20 µM. Simultaneously, glaucumolide B (172) also inhibited superoxide anion generation and elastase release in human neutrophils with the IC 50 values of 2.79 ± 0.32 and 3.97± 0.10 µM, respectively. The expression of iNOS and COX-2 proinflammatory proteins were reduced to 43.4 ± 5.0 and 6.0 ± 3.6% at 20 µM concentration of glaucumolide B (172). Ximaolide A (173) reduced the level of COX-2 expression to 22.0 ± 6.5% in LPS-treated macrophage cells at 20 µM [38].
An in vivo study unveiled that cembranoid 11-epi-sinulariolide acetate (33) could be a promising agent for the treatment of rheumatic arthritis as it significantly improved the clinical characteristics and the histopathologic features in AIA rat model. The expressions of osteoclast-related proteins, cathepsin K, MMP-9, TRAP, and TNF-a, in the ankle tissues of AIA rats were also reduced in the presence of 11-epi-sinulariolide acetate (33) [79].
The analgesic properties of flexibilide (34) were demonstrated in the rat model of carrageenan-induced acute inflammatory pain at a dose of 80 mg/kg [80]. Another study also revealed its anti-neuroinflammatory and analgesic effects in a rat chronic constriction injury model of neuropathic pain [31].
Excavatolide B (149), a briarane-type diterpene, not only demonstrated its anti-inflammatory effects in vitro and in vivo but also showed anti-rheumatic effects in adjuvant-induced arthritis (AIA) and type II collagen-induced arthritis (CIA) rat models. That evidence suggested that excavatolide B (149) could be a promising candidate for the treatment of rheumatic arthritis in humans [61,62].

Conclusion and Perspectives
Since 2002 up to the present, a total of 201 compounds, including 170 diterpenes, 8 biscembranoids, 20 steroids, and 3 other miscellaneous compounds have been isolated from different species of cultured-type soft corals belonging to the genera Sarcophyton, Sinularia, Briareum, Litophyton, Lobophytum, Klyxum, and Erythropodium. Obviously, diterpenes were the most common compounds found in these marine organisms, representing 84.6% of the isolates. They showed the most diverse structural variations with six subclasses, including eunicellin-based diterpene, cembrane-type diterpene, spatane-type diterpene, norcembranoidal diterpene, briarane-type diterpene, and aquariane-type diterpene. The second highest number of isolates belongs to steroids with 10%, followed by biscembranoids (4%). Most steroids and biscembranoids were discovered as previously unreported compounds (seventeen steroids and six biscembranes). Other quinones and α-tocopherol derivatives were also obtained from the cultured soft corals, which made up nearly 1.5% of the isolated compounds. Generally, novel compounds occupied nearly 70% of the isolates, showing anti-tumor and anti-inflammatory effects. Many isolated compounds displayed weak or moderate cytotoxicity, but it should be noted that only a limited panel of cancer cell lines was utilized to evaluate the cytotoxicity of the secondary metabolites, while there are hundreds of cell lines that were not used for the screening of the cytotoxic properties. Other biological properties were also observed in the isolated derivatives, including anti-acne, anti-rheumatic, antinociceptive, antibacterial activities, and neuroprotective effects.
According to our literature-based investigation, only pseudopterosins derived from the soft coral Pseudopterogorgia elisabethae were subjected to clinical trials for their antiinflammatory and analgesic effects and were successfully commercialized as a cosmetic product for human. It is remarkable that the supply of pseudopterosins is still derived from the exploitation of the wild-type soft coral in the Bahamas, which may lead to an upcoming supply problem [84]. Until now, no metabolite derived from aquaculture soft corals has been approved for clinical use as a therapeutic agent despite their pharmacological potential because the large-scale production of marine natural products for clinical trials has not been resolved effectively. The supply issue can be handled by the chemical synthesis of the potential compounds or aquaculture of the soft corals. Due to the structure complexity and unknown biosynthetic pathways of marine secondary metabolites, chemical synthesis may be more difficult and time-and resource-consuming than aquaculture methods. Consequently, coral aquaculture has attracted great interests regarding the discovery of advanced technologies for the optimization of soft coral mass production. So far, mariculture ("in situ" aquaculture-sea based) and captive breeding ("ex situ" aquaculture-aquarium based) have been the two main methods of coral aquaculture [6]. Moreover, biotechnological techniques have also been available to support coral aquaculture, including genetic manipulation and probiotics use. These advances can increase nutrition, mitigate coral diseases, and enhance the gene expression for the biosynthesis of the target metabolites to improve the production of potential compounds [85,86].
The anti-inflammatory effects of 11-epi-Sinulariolide acetate (33) were investigated in both in vitro and in vivo tests, which suggested that it is one of the tremendous candidates deserving further development as a therapeutic agent of rheumatic diseases and other inflammatory diseases [79]. In another investigation, a fraction derived from the ethyl acetate extract of the cultured Sinularia flexibilis displayed its anti-acne properties on an in vivo model-Cutibacterium acnes-induced Wistar rats. Concomitantly, the isolates derived from this fraction, including flexibilide (sinularin) (34), 11-Dehydrosinulariolide (41), and 3,4:8,11-Bisepoxy-7-acetoxycembra-15(17)-en-1,12-olide (43) also exhibited their antikeratinocyte proliferation and anti-inflammatory actions in various in vitro studies [33]. All these results support the development of marine natural products derived from the extract of aquaculture Sinularia flexibilis or the three bioactive secondary metabolites.
The productivity of the cultured types could be even much higher than those of the wild types. As a typical example of high-yield production of natural products from cultured soft corals, the cultured Briareum stechei afforded about 0.76 g/kg of excavatolide B, which was 3-fold higher than the yield of about 0.24 g/kg from the wild-type [61]. Aquaculture not only provides a sustainable resource for soft corals to investigate their secondary metabolites but may also stimulate the species to produce different compounds in comparison with the wild type corals. For instance, the cultured-type Klyxum simplex yielded klysimplexins A-H, while none of these compounds were found in the wildtype soft coral and simplexins A-I were found instead [24]. Another example is from the case of Sinularia flexibilis.The wild type produced flexibilisolide B and flexibilisin B while the cultivated soft coral yielded other two metabolites, flexibilisolide A (31) and flexibilisin A (32) [29]. These differences suggest that farming soft corals could increase the structure diversity of natural products and the possibility of discovering new bioactive substances as well.